DOI: 10.1002/cmdc.201300083

Synthesis and Hepatitis C Antiviral Activity of 1-Aminobenzyl-1H-indazole-3-carboxamide AnaloguesJing-Jing Shi,[a] Fei-Hong Ji,[b] Pei-Lan He,[b] Ya-xi Yang,[a] Wei Tang,[b] Jian-Ping Zuo,*[b] andYuan-Chao Li*[a]

Hepatitis C virus (HCV) is a small (+)-RNA virus classified in thegenus Hepaciviru of the family Flaviviridae.[1] Nearly 3 % of theglobal population (approximately 170 million) have been in-fected with HCV, while 3–4 million are newly infected eachyear. Untreated HCV infections can progress to liver fibrosis,steatosis, cirrhosis, and even hepatocellular carcinoma.[2] Sincethe discovery of the virus in 1989,[3] much effort has beenmade to find efficient antiviral therapies.[4] Currently, the stan-dard of care for patients with chronic hepatitis C is a combina-tion of pegylated interferon alpha (PEG-IFN-a) and ribavirin.[5]

However, this treatment regime is only effective for 40–60 % ofpeople infected with HCV genotype-1, which accounts for themajority of infections in the US, Europe, and Asia.[6] Further-more, the low success rate and high cost associated with thistreatment restrict its usage. Viral protease and polymerase in-hibitors are promising agents currently under development. In2011, two NS3A/4A protease inhibitors (boceprevir and telapre-vir) were approved by the US Food and Drug Administration(FDA) for the treatment of chronic hepatitis C infection. Incombination with PEG-IFN-a and ribavirin, both boceprevirand telaprevir produce a higher cure rate and shorter periodof treatment.[7] HCV NS5B polymerase is another attractivetarget for antiviral therapy.[8] Because of the high mutation andreplication rates of HCV,[9] it is unlikely that a preventive vac-cine will become available in the coming years, and as such,more effective anti-HCV drugs are urgently needed.

In previous studies,[10] we applied a HCV-infected system incombination with an automated high-throughput system toscreen an available compound library. We identified 1-(3-ami-nobenzyl)-1H-indazole-3-carboxamide (4 a) as a modest inhibi-tor of the HCV-infected system with an IC50 value of 5–10 mm

and low cytotoxicity against Huh 7.5.1 cells (CC50 >10 mm). Uti-lizing 4 a as a lead, we initiated a study to explore the struc-

ture–HCV antiviral activity relationshipsof 1-aminobenzyl-1H-indazole-3-carbox-amide derivatives.

The synthesis of compounds 5–7 isdepicted in Scheme 1. Compounds 2 a–c were obtained from commerciallyavailable 3-carboxylic indazole 1 by pre-paring the aroyl chloride and then re-acting it with the appropriate amine. Alkylation at position 1 of2 a with 1-(bromomethyl)-3-nitrobenzene was performed in

the presence of potassium carbonate to give nitro compound3 a ; compounds 3 b–c were synthesized analogously. After re-duction of the nitro groups with tin(II) chloride dihydrate, keyintermediate amines 4 a–c were obtained. Acylamides 5–7were subsequently accessed via acylation.

Acylamide compounds 10 a,b and 13 a,b were preparedfrom 1H-indazole-3-carboxamide 2 a, through benzylation, re-duction and acylation (Scheme 2).

Initial efforts were focused on evaluating different substitu-ents at various positions on the benzyl group (Table 1). Acyla-tion of the 3’-amino group of compound 4 a gives 5 a,b, andthese compounds exhibit significantly improved inhibitory ac-tivities, especially compound 5 b (IC50 = 0.125 mm), which has

Scheme 1. Synthesis of acylamide compounds 5 a–5 w, 6 a–6 b and 7. Re-agents and conditions : a) 1. SOCl2, 80 8C, 4 h, 2. R1NH2, THF, 0 8C, 5 h, 52–57 %; b) K2CO3, 1-(bromomethyl)-3-nitrobenzene, DMF, RT, 6 h, 85–87 %;c) SnCl2·2 H2O, THF, RT, overnight, 70–79 %; d) R2CO2H, HBTU, Et3N, DMF, RT,overnight, or R2COCl, Et3N, CH2Cl2, 0 8C, 5 h, 65–91 %.

[a] J.-J. Shi,+ Y.-x. Yang, Prof. Y.-C. LiDepartment of Medicinal ChemistryShanghai Institute of Materia Medica, Chinese Academy of Sciences555 Road Zu Chong Zhi, Zhangjiang Hi-Tech ParkShanghai 201203 (P. R. China)E-mail : ycli@mail.shcnc.ac.cn

[b] F.-H. Ji,+ P.-L. He, W. Tang, Prof. J.-P. ZuoLaboratory of ImmunopharmacologyState Key Laboratory of Drug ResearchShanghai Institute of Materia Medica, Chinese Academy of Sciences555 Road Zu Chong Zhi, Zhangjiang Hi-Tech ParkShanghai 201203 (P. R. China)E-mail : jpzuo@mail.shcnc.ac.cn

[+] These authors contributed equally to this work.

Supporting information for this article is available on the WWW underhttp://dx.doi.org/10.1002/cmdc.201300083.

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approximately a 40-fold higher bioactivity than 4 a. Further-more, 2’-amino (9, 10 a,b) and 4’-amino (12, 13 a, 13 b) ana-logues were found not to exhibit significant HCV inhibition,with the exception of compound 10 b that has a moderate IC50

value of 1.70 mm although this is significantly poorer than thesimilarly substituted analogue 5 a (IC50 = 1.17 mm). The findingsindicate that an amino group at position 3’ of the 1-aminoben-zyl-1H-indazole-3-carboxamide core is crucial for HCV inhibitoryactivity and that acylation of the amino group influences activi-ty.

Subsequently, the focus was shifted to different substitutionsaround the amino group in position 3’ of the 3-carboxamidemoiety. Aroyl analogues of 4 a (e.g. , 5 f and 5 g) were observedto exhibit improved antiviral activity (Table 2). However, alkano-

yl analogues 5 d,e and h weredevoid of HCV inhibitory activityat a compound concentrationof 0.5 mm. Additionally, phenyl-acetic analogue 5 c was foundto be a potent inhibitor (IC50 =

0.539 mm). These findings sug-gested that the presence of anaryl moiety in the acyl groupcould lead to improved bioac-tivity. Furthermore, a primaryamino group on the 3-carboxa-mide was found to be criticalfor activity, as substitutions(e.g. , 6 a, 6 b and 7) caused sig-nificant decrease in activity.

The results summarizedabove led to a study that tar-

geted further optimization of the inhibitory activity by varyingaroyl substituents at the 3’-amino group (Table 3). As anticipat-ed, the compounds of this series showed improved bioactivity.In particular, the analogues with an aroyl group substituted in

Scheme 2. Synthesis of acylamides A) 10 a–b and B) 13 a–b. Reagents and conditions : a) K2CO3, 1-(bromomethyl)-2-nitrobenzene (for 10 a–b) or 1-(bromomethyl)-4-nitrobenzene (for 13 a–b), DMF, RT, 6 h, 69–72 %; b) SnCl2·2 H2O,THF, RT, overnight, 70–79 %; c) RCOCl, Et3N, CH2Cl2, 0 8C, 5 h, 83–90 %.

Table 1. Structures and anti-HCV activities of 1-aminobenzyl-1H-indazole-3-carboxamide analogues determined in HCV-infected Huh 7.5.1 cells.

Compd R CC50 [mm] IC50[a] [mm] SI[b]

4 a 3’-A >10 5–10 >15 a 3’-B >10 1.17 >8.55 b 3’-C >10 0.125 >809 2’-A >10 >10 –10 a 2’-B >10 >10 –10 b 2’-C >10 1.70 >5.912 4’-A >10 >10 –13 a 4’-B >10 >10 –13 b 4’-C >10 >10 –

[a] Mycophenolic acid (MPA) was used as reference agent/positive con-trol ; in this assay, the IC50 value was 0.713 mm. [b] Selective index (SI) cal-culated as CC50/IC50 ; IC50 values were estimated by inhibition at five con-centrations; assays were performed in triplicate, and data represent simi-lar results.

Table 2. Structures and anti-HCV activities of 1-aminobenzyl-1H-indazole-3-carboxamide analogues determined in HCV-infected Huh 7.5.1 cells.

Compd R1 R2 CC50 [mm] IC50[c] [mm] SI[d]

5 c H >10 0.539 >18.5

5 d H CH3 92.1[a] 0[b] –

5 e H 86.0[a] 10.3[b] –

5 f H >10 0.674 >14.8

5 g H >10 0.267 >37.4

5 h H 89.2[a] 8.71[b] –

6 a Ph 96.2[a] 6.05[b] –

6 b Ph 94.8[a] 0[b] –

7 CH2COOMe >10 >10 –

[a] Cell viability (%) at 0.5 mm. [b] Percentage inhibition at 0.5 mm. [c] My-cophenolic acid (MPA) was used as reference agent/positive control ; inthis assay, the IC50 value was 0.807 mm. [d] Selective index (SI) calculatedas CC50/IC50 ; IC50 values were estimated by inhibition at five concentra-tions; assays were performed in triplicate, and data represent similar re-sults.

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the para position (e.g. , 5 n, 5 q, 5 t, 5 u and 5 v) exhibited signif-icantly increased activity compared with unsubstituted deriva-tive 5 b (IC50 = 0.125 mm). Specifically, compounds 5 n (IC50 =

0.013 mm) and 5 t (IC50 = 0.007 mm) were found to be the mostpotent inhibitors with low cytotoxicity. However, analogueswith substitutions at either the ortho or meta position (e.g. , 5 j,5 l, 5 m, 5 o, 5 p, 5 r and 5 s) did not show improved activity,with the exception of 5 i (IC50 = 0.035 mm). Furthermore, the an-tiviral potencies of compounds 5 i, 5 n, 5 q and 5 t were validat-ed by observation of their activities in an HCV replicon assay(Table 4). Analogues 5 n and 5 q possess superb anti-HCV activi-ty; both are efficient HCV replicon inhibitors, with EC50 valuesof 0.018 mm and 0.024 mm, respectively, coupled with low toxic-ity (CC50 = 20.67 mm and CC50 >100 mm, respectively).

In conclusion, starting from compound 4 a as an early leadinhibitor of HCV, extensive structure–activity relationships stud-ies around multiple positions on the core structure were per-formed. The results showed that the amino group of the 3-car-boxamide scaffold could not be substituted, and that com-pounds in which the benzamide with a para orientation at po-sition 3’ showed the best bioactivity. The results led to the dis-covery of two potent anti-HCV, compounds 5 n (IC50 = 0.013 mm

and EC50 = 0.018 mm ; SI = 1150) and 5 t (IC50 = 0.007 mm andEC50 = 0.024 mm ; SI> 4160). Given the potent biological activityand selectivity of compounds 5 n and 5 t, studies are plannedaimed at the further optimization of these series. Their acutetoxicities and pharmacokinetic properties will be conducted, aswell as mechanistic studies on this class of compounds againstHCV.

Experimental Section

General : Details of the instruments used are given in the Support-ing Information, along with characterization data for compounds2–13 not given below.

1H-Indazole-3-carboxamide (2 a): A mixture of 1H-indazole-3-car-boxylic acid (1) (3.24 g, 10 mmol) in SOCl2 (30 mL) was stirred atreflux for 4 h. Volatiles were removed by a stream of nitrogen fol-lowed by vacuum to give 1H-indazole-3-carbonyl chloride asa solid. The solid was redissolved in THF (50 mL), and the solutionwas cooled to 0 8C and then treated dropwise with 26 % NH3·H2O(8 mL). The mixture was stirred at RT for 5 h. Water (20 mL) wasadded, and the resultant precipitate was isolated by filtration,washed with water (30 mL) and THF (30 mL), and air dried to give1H-indazole-3-carboxamide (2 a) as a white solid (1.84 g, 57 %):1H NMR (300 MHz, [D6]DMSO): d= 13.49 (s, 1 H), 8.15 (d, J = 8.1 Hz,1 H), 7.69 (s, 1 H), 7.58 (d, J = 8.1 Hz, 1 H), 7.38 (t, J = 7.5 Hz, 1 H),7.30 (s, 1 H), 7.21 ppm (t, J = 7.5 Hz, 1 H); LC-MS (ESI): m/z (%):184.1(100) [M + Na]+ .

Compounds 2 b,c were prepared by the same procedure as de-scribed for 2 a.

1-(3-Nitrobenzyl)-1H-indazole-3-carboxamide (3 a): A mixture of2 a (1.61 g, 10 mmol), K2CO3 (2.76 g, 20 mmol) and 1-(bromometh-yl)-3-nitrobenzene (2.26 g, 10.5 mmol) in DMF (30 mL) was stirredat RT for 6 h. Water (50 mL) was then added, and the resultant pre-cipitate was isolated by filtration, washed with water (15 � 3 mL),and air dried to give 1-(3-nitrobenzyl)-1H-indazole-3-carboxamide(3 a) as a white solid (2.58 g, 87 %): 1H NMR (300 MHz, [D6]DMSO):

Table 3. Structures and anti-HCV activities of 1-aminobenzyl-1H-indazole-3-carboxamide analogues determined in HCV-infected Huh 7.5.1 cells.

Compd R CC50 [mm] IC50[a] [mm] SI[b]

5 i >10 0.035 >286

5 j >10 0.230 >43

5 k >10 0.115 >87

5 l >10 0.115 >87

5 m >10 0.238 >42

5 n >10 0.013 >769

5 o >10 0.603 >16

5 p >10 0.346 >29

5 q >10 0.035 >285

5 r >10 0.739 >14

5 s >10 0.213 >47

5 t >10 0.007 >1430

5 u 6.35 0.044 144

5 v >10 0.040 >250

5 w >10 0.110 >91

[a] Mycophenolic acid (MPA) was used as reference agent/positive con-trol ; in this assay, the IC50 value was 0.780 mm. [b] Selective index (SI) cal-culated as CC50/IC50 ; IC50 values were estimated by inhibition at five con-centrations; assays were performed in triplicate, and data represent simi-lar results.

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d= 8.16 (m, 3 H), 7.83 (d, J = 8.7 Hz, 1 H), 7.74 (s, 1 H), 7.63 (m, 2 H),7.44 (m, 2 H), 7.27 (t, J = 7.5 Hz, 1 H), 5.89 ppm (s, 2 H); LC-MS (ESI):m/z (%): 319.0 (100) [M + Na]+ .

Compounds 3 b,c, 8, 11 were prepared by the same procedure asdescribed for 3 a.

1-(3-Aminobenzyl)-1H-indazole-3-carboxamide (4 a): A mixture of3 a (400 mg, 1.35 mmol) and SnCl2·2H2O (1.22 g, 5.4 mmol) in THF(10 mL) was stirred at RT overnight, and then the solution wastreated with 5 m aq NaOH to pH 11, extracted with EtOAc (2 �50 mL), washed with brine (50 mL), dried (Na2SO4), filtered and con-centrated. Purification by chromatography (EtOAc/Pet. ether, 1:1)gave 4 a as a white solid (252 mg, 70 %): 1H NMR (300 MHz,[D6]DMSO): d= 8.17 (d, J = 8.1 Hz, 1 H), 7.67 (d, J = 8.6 Hz, 2 H), 7.40(m, 2 H), 7.23 (t, J = 7.5 Hz, 1 H), 6.93 (t, J = 7.5 Hz, 1 H), 6.40 (m, 2 H),6.32 (s, 1 H), 5.55 (s, 2 H), 5.10 ppm (br s, 2 H); 13C NMR (100 MHz,[D6]DMSO): d= 164.4, 149.4, 141.1, 137.9, 137.8, 129.6, 127.1, 123.0,122.8, 122.5, 115.1, 113.8, 112.7, 111.0, 53.2 ppm; LC-MS (ESI): m/z(%): 289.0 (100) [M + Na]+ .

Compounds 4 b,c, 9, 12 were prepared by the same procedure asdescribed for 4 a.

1-(3-(Cyclopropanecarboxamido)benzyl)-1H-indazole-3-carboxa-mide (5 a): A solution of 4 a (27 mg, 0.1 mmol) and Et3N (0.028 mL,0.2 mmol) in dry CH2Cl2 (1 mL) at 0 8C under Ar was treated withcyclopropanecarbonyl chloride (0.010 mL, 0.11 mmol). The mixturewas stirred at 0 8C for 30 min. The solvent was removed in vacuoto give the crude product, which was purified by chromatography(CH2Cl2/MeOH, 50:1) to give 5 a as a white solid (29 mg, 88 %):1H NMR (300 MHz, [D6]DMSO): d= 10.14 (s, 1 H), 8.18 (d, J = 8.3 Hz,1 H), 7.69 (m, 2 H), 7.51 (d, J = 8.4 Hz, 1 H), 7.41 (m, 3 H), 7.23 (dd,J = 15.6, 7.6 Hz, 2 H), 6.90 (d, J = 7.2 Hz, 1 H), 5.68 (s, 2 H), 1.69 (m,1 H), 0.73 ppm (m, 4 H); 13C NMR (100 MHz, [D6]DMSO): d= 171.7,163.8, 140.7, 139.6, 137.6, 137.4, 129.0, 126.7, 122.5, 122.4, 122.1,121.8, 118.3, 117.5, 110.4, 52.4, 14.5, 7.16 ppm (2C); LC-MS (ESI): m/z (%): 357.0 (100) [M + Na]+ .

Compounds 5 b–w, 6 a,b, 7, 10 a,b, 13 a,b were prepared by thesame procedure as described for 5 a.

Acknowledgements

This work was supported by a National Science and TechnologyMajor Project (China): “Chemical Innovative Drug Research andDevelopment System” (grant no. 2012ZX09301-001).

Keywords: antiviral agents · carboxamides · hepatitis C ·organic synthesis · structure–activity relationships

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Table 4. Results of the HCV replicon assays for compounds 5 i, 5 n, 5 q,and 5 t.

Compd CC50 [mm] EC50 [mm] SI[a]

5 i 10.31 0.032 3225 n 20.67 0.018 11505 q 28.42 0.182 1565 t >100 0.024 >4160

[a] Selective index (SI) calculated as CC50/EC50 ; EC50 values were estimatedby inhibition at five concentrations; assays were performed in triplicate,and data represent similar results.

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COMMUNICATIONS

J.-J. Shi, F.-H. Ji, P.-L. He, Y.-x. Yang,W. Tang, J.-P. Zuo,* Y.-C. Li*

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Synthesis and Hepatitis C AntiviralActivity of 1-Aminobenzyl-1H-indazole-3-carboxamide Analogues

Fighting HCV: Two potent antiviral ana-logues were developed from a previous-ly identified lead as novel agentsagainst hepatitis C virus. Their potencyand selectivity (5 n : IC50 = 0.013 mm andEC50 = 0.018 mm ; 5 t : IC50 = 0.007 mm andEC50 = 0.024 mm) make them good can-didates for further development as anti-viral agents.

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